|
| Status |
Public on Dec 12, 2020 |
| Title |
HT-29_KO_MIEN1_3 |
| Sample type |
SRA |
| |
|
| Source name |
HT-29 cells KO MIEN1 replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
genotype: HT-29-pMIEN1-X (MIEN1 KO)
cell line: HT-29
|
| Treatment protocol |
HT-29 wild type (HT29-WT) cells were cultured and electroporated with a DNA mixture of plasmids expressing C1486_MIEN1_5'gRNA- 1, C1486_MIEN1_3'gRNA-1 and cas9 gene. Two days after transfection the cells were selected by 1.0-1.5 ug/ml of Puromycin for a period of 48 hours. A small portion of the cell culture, presumably containing the mixed population, was subjected to genotype analysis. The mixed culture was diluted to less than 1 cell/200ul culture media and dispersed into each well of a 96-well plate. The cells were allowed to grow for 15 to 20 days. Cells derived from such single cell cloning process were subjected to genotype analysis. The positive clones for the desired mutation were cultured-expanded and a portion was used to confirm the predicted genotype. Genotyping was performed by using genomic DNA from a single cell clone. A ~400bp DNA fragment was amplified using specific primers and subjected to direct sequencing
|
| Growth protocol |
HT-29 cells (ATCC, Manassas, VA , USA) were seeded at 50.000 cells per well in 24-well culture dishes in Dulbecco´s Modified Eagle´s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
HT-29 WT and HT-29-pMIEN1-X cells were cultured in 24-well dishes as mentioned above. Total RNA was extracted from cells (80.000 cells per well) using TRIzol and the Ambion Purelink RNA mini kit (Invitrogen). Equal amounts of RNA from four dishes were pooled (biological replicates, BR). Four independent BR were obtained from WT (WT1-WT4) and mutant conditions (Mut1-Mut4). For eukaryotic transcriptome library constructing the mRNA was selected using poly–T oligo–attached magnetic beads.
Poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads and fragmented using divalent cations under elevated temperature. First strand cDNA was synthetized using reverse transcriptase and random hexamer primer. Then second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments of preferentially ~150-200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the products were enriched with PCR and purified to create the cDNA library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Raw reads were filtered to remove adapters and low quality reads (Qscore > 50% bases <= 5 or Ns > 10% bases).
Paired-end reads were aligned to human genome build GRCh38-Ensembl using TopHat2 v2.0.9(Kim et al. 2013).
Gene expression was quantified using HTSeq v0.6.1(Anders, Pyl, and Huber 2015) using the union mode (-m union)
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include read counts data for each sample.
|
| |
|
| Submission date |
Dec 12, 2018 |
| Last update date |
Dec 13, 2020 |
| Contact name |
Oscar Javier Ortega-Recalde |
| E-mail(s) |
ojavieror@gmail.com
|
| Organization name |
University of Otago
|
| Department |
Anatomy
|
| Street address |
60 Hanover Street
|
| City |
Dunedin |
| State/province |
Otago |
| ZIP/Postal code |
9016 |
| Country |
New Zealand |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE123734 |
Functional dissection of MIEN1 trans-regulation provides new evidence for colorectal cancer genome editing-based therapeutics |
|
| Relations |
| BioSample |
SAMN10586094 |
| SRA |
SRX5126890 |
